A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo

Taha, T. Y.; Suryawanshi, R. K.; Chen, I. P.; Correy, G. J.; O'Leary, P. C.; Jogalekar, M. P.; McCavitt-Malvido, M.; Diolaiti, M.; Kimmerly, G. R.; Tsou, C.-L.; Martinez-Sobrido, L.; Krogan, N. J.; Ashworth, A.; Fraser, J. S.; Ott, M.

doi:10.1101/2023.04.18.537104

bioRxiv · 10.1101/2023.04.18.537104

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo

Taha, T. Y.·Suryawanshi, R. K.·Chen, I. P.·Correy, G. J.·O'Leary, P. C.·Jogalekar, M. P.·McCavitt-Malvido, M.·Diolaiti, M.·Kimmerly, G. R.·Tsou, C.-L.·Martinez-Sobrido, L.·Krogan, N. J.·Ashworth, A.·Fraser, J. S.·Ott, M.

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Abstract

Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by [~]10-fold, mutations to aspartic acid (N40D) reduced activity by [~]100-fold relative to wildtype. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.

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Taha, T. Y., Suryawanshi, R. K., Chen, I. P., Correy, G. J., O'Leary, P. C., Jogalekar, M. P., McCavitt-Malvido, M., Diolaiti, M., Kimmerly, G. R., Tsou, C.-L., Martinez-Sobrido, L., Krogan, N. J., Ashworth, A., Fraser, J. S., Ott, M.. 2023-04-18. A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo. https://doi.org/10.1101/2023.04.18.537104

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